Pneumococcus (Streptococcus pneumoniae) is a Gram-positive bacterium belonging to the family Streptococcaceae, within the order Lactobacillus, and it causes diseases such as pneumonia, bacteremia, otitis media, and meningitis in humans. Cells of pneumococcal serotypes have a capsule which is a polysaccharide coating surrounding each cell. This capsule interferes with phagocytosis by preventing antibodies from attaching to the bacterial cells. The cells have been categorized into over 90 serotypes based on immunological characteristics, some of which are reported to cause invasive diseases. A total of 37 serotypes were identified from invasive Streptococcus pneumoniae collected from 1996 to 2008, and the main serotypes, 19F>23F>19A>6A>3>9V>6B frequent in this order, account for a majority of 53.4%. Capsular polysaccharides of these pneumococcal serotypes have been used in the production of vaccines such as polysaccharide vaccines and polysaccharide-protein conjugate vaccines.
Capsular polysaccharides used in the production of polysaccharide vaccines and polysaccharide-protein conjugate vaccines are obtained from culture broths of bacterial cells producing respective serotypes. Each strain is cultured under optimal temperature, pH and agitation until it reaches a maximum density. To obtain pneumococcal capsular polysaccharides that are not secreted outside the cytoplasm, cell lysis is carried out using a lysing agent such as sodium deoxycholate (DOC). When cells are lysed, various proteins and nucleic acids of the strain are released, together with capsular polysaccharides, and they must be removed by a post-culture purification process. To minimize the risk of adverse events by any substance other than an antigen after vaccination, there are strict specifications for protein and nucleic acid contents. For instance, in the case of Prevnar 7™, protein and nucleic acid contents in each serotype satisfy the specifications of 2-5% or less and 1-2% or less on a dry weight basis, respectively.
International Patent Publication No. WO 2006/110352 discloses a method of producing capsular polysaccharides comprising cultivating of Streptococcus pneumonia, cell lysis, protein precipitation by acidification of the cell lysate to a pH of less than 5.5, incubation without agitation, and centrifugation and/or filtration. Further, International Patent Publication No. WO 2008/118752 discloses a method of producing capsular polysaccharides by performing ultrafiltration and diafiltration of a cell lysate followed by a protein precipitation process by acidification. However, all these related production methods require the protein precipitation process by acidification with a pH adjuster, which makes the overall process complicated and also causes modification of capsular polysaccharide and generation of harmful substances.